Persister populations of Mycobacterium tuberculosis in sputum that grow in liquid but not on solid culture media

OBJECTIVES : Can the characteristics of persisters in cultures of Mycobacterium tuberculosis also be found in bacilli from the sputum of pulmonary tuberculosis patients? The objective of this study was to explore whether the ability of persisters to grow in liquid but not on solid culture media, as in 100 day static cultures, can also be found in bacilli in sputum. METHODS : Serial dilutions of homogenized sputum obtained from patients before or during the first week of treatment were inoculated into broths to estimate the probable number of organisms and onto plates to give colony counts. RESULTS : Cultures in broths grew slowly to reach a maximal count at 12 weeks of probable numbers about 10-fold higher than the colony counts on plates, which did not grow beyond the initial count at 3-4 weeks. No such excess growth in liquid medium was found with control log-phase cultures. CONCLUSIONS : About 90% of the bacilli in sputum are persisters that can grow in liquid media but not on solid plates.Sputum specimens were examined as part of the quality assurance programme of the Oflotub clinical trial,14 financially supported by a grant (ICA4-CT2002-10057) from the European Commission and another (OD/TS-04-00291) from the WHO special programme for research and training in tropical diseases (TDR).http://jac.oxfordjournals.orghb201

Mycobacterium tuberculosis from chronic murine infections that grows in liquid but not on solid medium

BACKGROUND: Old, stationary cultures of Mycobacterium tuberculosis contain a majority of bacteria that can grow in broth cultures but cannot grow on solid medium plates. These may be in a non-replicating, dormant growth phase. We hypothesised that a similar population might be present in chronic, murine tuberculosis. METHODS: Estimates of the numbers of viable M. tuberculosis, strain H37Rv, in the spleens and lungs of mice in a 7-day acute infection and in a 10-month chronic infection were made by conventional plate counts and, as broth counts, by noting presence or absence of growth in serial replicate dilutions in liquid medium. RESULTS: Plate and broth counts in 6 mice gave similar mean values in the acute infection, 7 days after infection. However, the broth counts were much higher in 36 mice with a chronic infection at 10 months. Broth counts averaged 5.290 log(10 )cfu /organ from spleens and 5.523 log(10 )cfu/organ from lungs, while plate counts were 3.858 log(10 )cfu/organ from spleens and 3.662 log(10 )cfu/organ from lungs, indicating that the total bacterial population contained only 3.7% bacilli in spleens and 1.4% bacilli in lungs, capable of growth on plates. CONCLUSION: The proportion growing on plates might be a measure of the "dormancy" of the bacilli equally applicable to cultural and animal models

A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated with bacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing

Four-Month High-Dose Rifampicin Regimens for Pulmonary Tuberculosis

BACKGROUND Shorter but effective tuberculosis treatment regimens would be of value to the tuberculosis treatment community. High-dose rifampicin has been associated with more rapid and secure lung sterilization and may enable shorter tuberculosis treatment regimens. METHODS We randomly assigned adults who were given a diagnosis of rifampicin-susceptible pulmonary tuberculosis to a 6-month control regimen, a similar 4-month regimen of rifampicin at 1200 mg/d (study regimen 1 [SR1]), or a 4-month regimen of rifampicin at 1800 mg/d (study regimen 2 [SR2]). Sputum specimens were collected at regular intervals. The primary end point was a composite of treatment failure and relapse in participants who were sputum smear positive at baseline. The noninferiority margin was 8 percentage points. Using a sequence of ordered hypotheses, noninferiority of SR2 was tested first. RESULTS Between January 2017 and December 2020, 672 patients were enrolled in six countries, including 191 in the control group, 192 in the SR1 group, and 195 in the SR2 group. Noninferiority was not shown. Favorable responses rates were 93, 90, and 87% in the control, SR1, and SR2 groups, respectively, for a country-adjusted absolute risk difference of 6.3 percentage points (90% confidence interval, 1.1 to 11.5) comparing SR2 with the control group. The proportions of participants experiencing a grade 3 or 4 adverse event were 4.0, 4.5, and 4.4% in the control, SR1, and SR2 groups, respectively. CONCLUSIONS Four-month high-dose rifampicin regimens did not have dose-limiting toxicities or side effects but failed to meet noninferiority criteria compared with the standard 6-month control regimen for treatment of pulmonary tuberculosis. (Funded by the MRC/Wellcome Trust/DFID Joint Global Health Trials Scheme; ClinicalTrials.gov number, NCT02581527.